Intrathymic inoculation with foreign antigen has proven to be an effective means to induce a durable state of central immunological tolerance. Despite the dramatic success of this approach in experimental animals, to date clinical application is lacking; we believe this is in large part a consequence of our incomplete understanding of the mechanisms involved in the induction and maintenance of the tolerant state. In this proposal, we will dissect the mechanisms of acquired central tolerance using a line of T cell receptor transgenic mice (TS1) in which a high percentage of CD4 T cells have class II restricted specificity for a dominant epitope (Si) of the viral hemagglutinin (HA) molecule. Transgenic HA specific T cells are readily detected using the clonotype specific antibody 6.5. Although many TcR transgenic mouse lines have been reported, few have proven useful in transplantation models. In sharp contrast, the TS1 line is uniquely suited for the study of graft rejection and transplantation tolerance. This is due in part to the availability of a number of lines of transgenic mice with diffuse tissue expression of the HA transgene that can serve as donors for grafts bearing the foreign neo-xmnor antigen HA. We have confirmed in preliminary studies that TSI recipients reject grafts from otherwise syngeneic donors that express an HA transgene. Graft rejection is dependent on 6.5+ T cells, and these cells are sufficient to mediate HA+ graft rejection upon transfer to immunodeficient hosts. HA+ graft rejection can be prevented in IS 1 recipients by prior intrathymic inoculation of the iminunodominant peptide of the HA molecule. Using TS1 mice, we will explore the mechanisms involved in the induction of acquired central tolerance and attempt to clarify the complex interaction between thymic and peripheral immune compartments. As a prelude to tolerance experiments, in Aim 1 we will characterize further the rejection of HA expressing grafts by TS 1 mice and determine the relative involvement of CD4 and CD8 T cells. In aim II, we will explore our hypothesis that acquired central tolerance is not mediated solely by passive mechanisms of clonal deletion and/or anergy. We will focus our studies on whether an antigen injected thymus exports regulatory cells that modulate the response of naive peripheral T cells. We are particularly interested in the role of a recently described population of anergic/regulatory T cells (CD4+, CD25+) that appear to be critical mediators of self-tolerance to tissue antigens. Discerning the function of these cells in the model of acquired central tolerance in TS 1 mice is an important goal of the experiments in Aim II. Using cells from tolerant mice, we will also determine whether rejection of grafts carrying traditional histocompatibility antigens in addition to HA can be prevented by linked suppression. In the final Aim, we will probe the factors that determine whether developing I cells undergo thymic deletion vs. acquisition of anergic/ regulatory T cell function. Specifically, we will determine if the decision to delete or anergize can be impacted on by varying either the avidity of the interaction between antigen and the transgenic TcR of developing thymocytes, or whether the type of thymic APC that presents antigen injected into the thymus dictates a particular pathway of tolerance.